Reduced gap junction (GJ) coupling and muscle loss caused by unregulated opening of Cx43 hemichannels (HCs) in the acute phase following a myocardial infarction are thought to contribute to formation of an arrhythmic substrate. In the previous period, we showed that a mimetic peptide corresponding to the PDZ-binding domain of Cx43 (?CT1) preserved action potential conduction properties, reduced arrhythmias and improved contractile function following myocardial injury. We further demonstrated that the mechanism of ?CT1 involved inhibition of interaction between Cx43 and ZO-1 in a membrane microdomain adjacent to the GJ - the perinexus. Inhibition of Cx43:ZO-1 interaction prompted release of HCs from the perinexus and their sequestration into GJs- thus inhibiting hemichannel activity. ?CT1 was also found to increase phosphorylation at a PKC? site on Cx43 at serine 368 (S368). In new data, we show that ?CT1 prompts this Cx43 modification via a novel mechanism that involves induction of distinct phosphorylated nanodomains within GJs. Moreover, in a collaborative study we determined that ?CT1 binds to the Cx43 loop domain. Importantly, the minimal sequence required for this interaction corresponds to that of Gap19 - another Cx43 mimetic peptide reported to inhibit hemichannels. Based on the newly identified interaction between Gap19 and the Cx43 PDZ-binding domain we propose the hypothesis that the mechanism of Gap19 also involves targeting of Cx43:ZO-1 interaction and is complementary to that of ?CT1. Namely, that Gap19 interacts with the Cx43 CT obstructing its engagement with the PDZ2 domain of ZO-1 - as opposed to ?CT1, which binds to PDZ2 thereby blocking Cx43 CT interaction. In this renewal we aim to: 1) Identify the molecular determinants of ?CT1-induced phosphorylation of Cx43 at serine 368 - a post-translational modification that inhibits HC activity and determine the degree to which S368 phosphorylation explains HC inhibition by ?CT1 and Gap19; 2) Determine whether Cx43 HC inhibitors spare heart muscle and prevent formation of an arrhythmic substrate if given in the acute hours following myocardial infarction; and 3) Determine whether a common aspect of ?CT1 and Gap19 mechanism is that both peptides prompt selective inhibition of hemichannel activity via disruption of Cx43:ZO-1 interactions in the perinexus. The tools used will include state-of-the- art super-resolution microscopy and functional MRI approaches, new rationally designed ?CT1 variants and novel cell lines and transgenic mouse models expressing a phospho-null S368A mutation. Progress from the previous funding period included publication of results from two Phase II clinical trials on ?CT1. The studies proposed herein will provide mechanistic understanding of new drugs targeting connexins and studies in animals that provide a necessary prelude to clinical testing of these drugs in humans as muscle-sparing and anti-arrhythmic therapies in the critical hours following a myocardial infarction.